DESCRIPTION (Verbatim from the Applicant's Abstract): The midline of the CNS is an interesting boundary for neuronal growth cones and provides an ideal system for dissecting molecular mechanisms of axon guidance. In vertebrates and invertebrates, the CNS midline produces both attractive and repulsive guidance cues and regulates the ability of axons to cross or not to cross. Our studies focus primarily on the Drosophila embryonic CNS where a powerful combination of genetic, molecular, and cellular approaches can be utilized to address these fundamental issues of axon guidance. This application addresses two key regulators of guidance at the midline: Commissureless (Comm) and Roundabout (Robo). Robo is the receptor for the midline chemorepellent Slit and functions to prevent axons from crossing the midline boundary. Comm encodes a novel transmembrane protein that is required for axons to cross the midline. Comm functions, at least in part, by regulating the surface accumulation of the Robo receptor. Comm and Robo form a coimmunoprecipitable complex that somehow leads to the endocytosis or membrane clearance of the Robo receptor. We have begun to map regions of Comm that are required for this effect on Robo and find that a Comm protein consisting of the transmembrane domain and the first 97 amino acids of the cytoplasmic domain is necessary and sufficient for this activity. The experiments outlined in this application are designed to test and extend our hypotheses about Comm function. We will further characterize the Comm-Robo interaction, defining amino acids required for this interaction . These structure-function studies will provide insights into the molecular mechanisms by which Comm regulates Robo. We will define where Comm activity is required during embryonic development. Complexities regarding Comm mRNA and protein distribution make this a critical issue to resolve. Comm is required for proper neuromuscular synatogenesis; as a first step in understanding this aspect of Comm function we will determine what regions of Comm are required for synaptogenesis. Finally, we propose to identify other components of the Comm and Robo pathways by using genetic screens to isolate enhancers and suppressors of specific comm mutant phenotypes. While inhibitory guidance receptors like Robo are essential for neural development, their presence at latter stages is problematic for axon regeneration. Understanding how these proteins function and are regulated is essential for the development of treatments for the devastating consequences of spinal cord injury.